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Microengineered cultures containing human hepatic stellate cells and hepatocytes for drug development
Integrative Biology ( IF 1.5 ) Pub Date : 2017-07-03 00:00:00 , DOI: 10.1039/c7ib00027h
Matthew D. Davidson 1, 2, 3, 4, 5 , David A. Kukla 4, 5, 6, 7 , Salman R. Khetani 1, 2, 3, 4, 5
Affiliation  

In non-alcoholic steatohepatitis (NASH), hepatic stellate cells (HSC) differentiate into myofibroblast-like cells that cause fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. Thus, modeling interactions between activated HSCs and hepatocytes in vitro can aid in the development of anti-NASH/fibrosis therapeutics and lead to a better understanding of disease progression. Species-specific differences in drug metabolism and disease pathways now necessitate the supplementation of animal studies with data acquired using human liver models; however, current models do not adequately model the negative effects of primary human activated HSCs on the phenotype of otherwise well-differentiated primary human hepatocytes (PHHs) as in vivo. Therefore, here we first determined the long-term effects of primary human activated HSCs on PHH phenotype in a micropatterned co-culture (MPCC) platform while using 3T3-J2 murine embryonic fibroblasts as the control cell type since it has been shown previously to stabilize PHH functions for 4–6 weeks. We found that HSCs were not able to stabilize the PHH phenotype to the same magnitude and longevity as the fibroblasts, which subsequently inspired the development of a micropatterned tri-culture (MPTC) platform in which (a) micropatterned PHHs were functionally stabilized using fibroblasts, and (b) the PHH phenotype was modulated by culturing HSCs within the fibroblast monolayer at physiologically-relevant ratios with PHHs. Transwell inserts containing HSCs were placed atop MPCCs containing fibroblasts to confirm the effects of paracrine signaling between PHHs and HSCs. We found that while albumin and urea secretions were relatively similar in MPTCs and MPCCs (suggesting well-differentiated PHHs), increasing HSC numbers within MPTCs downregulated hepatic cytochrome-P450 (2A6, 3A4) and transporter activities, and caused steatosis over 2 weeks. Furthermore, MPTCs secreted higher levels of pro-inflammatory interleukin-6 (IL-6) cytokine and C-reactive protein (CRP) than MPCCs. Treatment of MPCCs with HSC-conditioned culture medium confirmed that HSC secretions mediate the altered phenotype of PHHs observed in MPTCs, partly via IL-6 signaling. Lastly, we found that NADPH oxidase (NOX) inhibition and farnesoid X receptor (FXR) activation using clinically relevant drugs alleviated hepatic dysfunctions in MPTCs. In conclusion, MPTCs recapitulate symptoms of NASH- and early fibrosis-like dysfunctions in PHHs and have utility for drug discovery in this space.

中文翻译:

包含人类肝星状细胞和肝细胞的微工程培养物,用于药物开发

在非酒精性脂肪性肝炎(NASH)中,肝星状细胞(HSC)分化为引起纤维化的成肌纤维母细胞样细胞,这使患者更容易患肝硬化和肝细胞癌。因此,在体外模拟活化的HSC与肝细胞之间的相互作用可以帮助开发抗NASH /纤维化疗法,并有助于更好地了解疾病的进展。现在,由于药物代谢和疾病途径的物种特异性差异,需要对动物研究进行补充,这些研究需要使用人类肝脏模型获得的数据。然而,目前的模型未能充分模拟原代人激活的HSC对体内分化良好的原代人肝细胞(PHH)表型的负面影响。因此,在此我们首先确定了人类活化的HSC在微模式共培养(MPCC)平台中对PHH表型的长期影响,同时使用3T3-J2鼠胚胎成纤维细胞作为对照细胞类型,因为先前已经证明它可以稳定PHH的功能持续4到6周。我们发现,HSC不能将PHH表型稳定在与成纤维细胞相同的大小和寿命,这随后激发了微模式三培养(MPTC)平台的发展,在该平台中(a)使用成纤维细胞功能性稳定了微模式PHH, (b)通过在成纤维细胞单层内以与PHHs生理相关的比例培养HSC来调节PHH表型。将含有HSC的Transwell插入片段置于含有成纤维细胞的MPCC上,以确认PHH和HSC之间的旁分泌信号传导的作用。我们发现,虽然MPTC和MPCC中白蛋白和尿素的分泌相对相似(暗示PHHs高度分化),但MPTC中HSC数量的增加下调了肝细胞色素P450(2A6、3A4)和转运蛋白的活性,并在2周内引起脂肪变性。此外,MPTC比MPCC分泌更高水平的促炎性白介素6(IL-6)细胞因子和C反应蛋白(CRP)。用HSC条件培养基处理MPCC证实,HSC分泌介导了在MPTC中观察到的PHH表型改变。我们发现,虽然MPTC和MPCC中白蛋白和尿素的分泌相对相似(暗示PHHs高度分化),但MPTC中HSC数量的增加下调了肝细胞色素P450(2A6、3A4)和转运蛋白的活性,并在2周内引起脂肪变性。此外,MPTC比MPCC分泌更高水平的促炎性白介素6(IL-6)细胞因子和C反应蛋白(CRP)。用HSC条件培养基处理MPCC证实,HSC分泌介导了在MPTC中观察到的PHH表型改变。我们发现,虽然MPTC和MPCC中的白蛋白和尿素分泌相对相似(建议高度分化的PHH),但MPTC中HSC数量的增加下调了肝细胞色素P450(2A6、3A4)和转运蛋白的活性,并在2周内引起脂肪变性。此外,MPTC比MPCC分泌更高水平的促炎性白介素6(IL-6)细胞因子和C反应蛋白(CRP)。用HSC条件培养基处理MPCC证实,HSC分泌介导了在MPTC中观察到的PHH表型改变。通过IL-6信号传导。最后,我们发现使用临床相关药物抑制NADPH氧化酶(NOX)和法尼醇X受体(FXR)的活化可减轻MPTC中的肝功能障碍。总之,MPTC概括了PHH中NASH和早期纤维化样功能障碍的症状,并在该领域发现药物有用。
更新日期:2017-08-15
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